Sickle cell disease (SCD) is one of the most prevalent monogenic diseases in Europe. A single amino acid substitution in the beta-globin chain of the adult hemoglobin (Hb) drives red blood cell sickling and multi-organ damage. The clinical severity of SCD is alleviated by the co-inheritance of mutations causing expression of fetal gamma-globin in adult life ? a condition termed hereditary persistence of fetal hemoglobin (HPFH). Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option for SCD patients. To this end, genome editing approaches based on the use of site-specific nucleases or, more recently, base editors have been explored by many groups, including teams in our consortium. These approaches either correct the single point mutation causing SCD or reactivate fetal gamma-globin expression by mimicking HPFH mutations. On the other hand, (pre)clinical data from SCD patients or SCD mouse models, as well as preliminary data from our labs suggest that SCD HSPCs are characterized by a high mutational burden, oxidative stress and expression of inflammatory genes. This can alter HSPC properties as well as their interactions within the bone marrow niche. In the context of gene therapy, it is essential to understand the mechanisms underlying SCD HSPC dysfunction and assess the impact of genome editing approaches on SCD HSPCs. In this proposal, we have assembled a multidisciplinary team to: (i) understand the molecular and cellular mechanisms underlying SCD HSPC autonomous and non-cell-autonomous dysfunctions and (ii) evaluate the impact of established and novel genome editing approaches on SCD HSPC properties and genome integrity.
In particular, we will compare established and novel therapeutic approaches for SCD based on genome editing. In particular, we will test three therapeutic strategies:
1. A CRISPR/Cas9 nuclease approach to target the HBG promoters and disrupt the LRF BS by NHEJ to reactivate HbF.
2. A CRISPR/Cas9 approach to revert the SCD mutation by HDR.
3. A base editing approach either to generate HPFH mutations in the HBG promoters or to revert the SCD mutation by generating the Makassar β-globin variant. We recently developed a base editing approach to introduce HPFH mutations that either disrupt the LRF repressor BS using CBE or generate a KLF1 activator BS using ABE.Furthermore we have established a highly efficient ABE-based strategy to generate the Makassar variant in SCD hHSPCs.
This study will lay the foundation of an improved gene therapy strategy to treat SCD and provide best practice tools and protocols for genome editing-based therapies in HSPCs.
